The interactions of T lymphocytes with antigen presenting cells (APC) are central to the generation of an immune response to foreign pathogens, transplanted organs, and self-antigens in the setting of autoimmune disease. The specificity of these interactions is provided by recognition of an antigen on the APC by the T cell antigen receptor (TCR). TCR engagement alone does not generally lead to full T cell activation, but instead may lead to T cell unresponsiveness (anergy). A successful immune response requires additional interactions between the T cell and APC. These so-called "costimulatory" interactions thus determine the outcome of TCR engagement; that is, whether this engagement activates or inactivates subsequent immune responses. It is now clear that a key T cell costimulatory signal is provided by interaction of CD28 receptors on T cells with B7 counter-receptors on APC. Like CD28, CTLA-4 is a second receptor for B7 molecules; however, it has a higher avidity for B7 molecules than does CD28. Interference with CD28/CTLA-4:B7 interactions may thus represent a novel approach to regulating the interactions of the cells that have these receptors. The use of a native soluble form of CTLA-4 may thus serve as a immunoregulatory agent.
Other investigators have used a CTLA-4 fusion protein (the extracellular domain of the membrane form of CTLA-4 fused to a human immunoglobulin heavy chain gene) called CTLA-4Ig to regulate CD28/CTLA-4:B7 interactions. Potential limitations of this approach include unnatural tissue distribution of this entity as well as unnatural pharmacokinetics of the molecule when compared to the molecule described in the present invention.
CTLA-4 is a lymphocyte cell surface receptor originally discovered in a search for molecules having a role in T cell cytotoxicity. Although originally identified as a cytolytic T cell-associated molecule, CTLA-4 expression has been detected in both activated CD4 and CD8 T-cell populations as well as B-cells with the use of monoclonal antibodies. CTLA-4 is a member of the immunoglobulin superfamily and is highly homologous to another T cell surface receptor, CD28. Both CD28 and CLTA-4 bind the same ligands; namely, members of the B7 family of molecules expressed on antigen presenting cells. Expression of CTLA-4 is highly activation dependent. With activated T cells, maximal CTLA-4 protein expression is .about.2-3% of CD28. Recombinant forms of CTLA-4 bind CD80 (B7.1) and CD86 (B7.2) with higher avidity (&gt;20-fold) than soluble CD28. Thus, CTLA-4 is a high avidity, low abundance receptor for B7 molecules.
Much of the study of the interactions of CTLA-4 and B7 counter-receptors have relied on the use of recombinant soluble form of CTLA-4 (called CTLA4-Ig). CTLA4-Ig is a fusion protein consisting of the Ig-like extracellular domain of CTLA-4 and the human IgG heavy chain constant region gene. The presence of the IgG chain permits secretion of the recombinant protein from transfected cells, as well as enhancing purification of the molecule. Numerous reports have documented the usefulness of CTLA4-Ig as an immunoregulatory agent. For example, CTLA4-Ig inhibits alloreactivity in the content of both the mixed leukocyte reaction and in vivo in experimental cardiac allografts and pancreatic islet xenografts. CTLA-Ig is also reported to modify the course of several animal models of human disease including murine lupus, experimental autoimmune encephalomyelitis, allergic contact dermatitis, and autoimmune and anti-glomerular basement membrane glomerulonephritis. In addition, CTLA4-Ig also inhibits cellular and humoral responses to conventional antigens such as keyhole-limpet hemocyanin, sheep red blood cells, and pigeon cytochrome c.
It would be extremely useful to obtain expression of a native soluble protein product of the CTLA-4 gene. The soluble product could then be used to regulate T cell and/or B cell responses in vivo to treat pathological conditions.
The molecular cloning and the nucleotide sequence of the full length rat CTLA-4 gene has been reported. In addition to the expected target CTLA-4 transcript, distinct smaller amplification products were also observed following reverse transcriptase polymerase chain reaction (RT-PCR) of a variety of lymphoid tissues from man, mouse, and rat. The nucleotide and the predicted amino acid sequence of these alternative transcripts are described below. See Sequence ID Nos. 1-6. In addition, it will be shown that the variant transcript is most abundant in cells from bone marrow, blood, and lymph node; whereas, the transmembrane form of the molecule is the predominant, and perhaps the only species expressed in the adult thymus.